Glycerol shock or chloroquine? - CaPO4 Transfection Optimization (Aug/18/2008 )

Hello all!

I have another question regarding calcium phosphate transfections. I am planning to do another this week, but I would like to compare standard CaPO4 transfection efficiencies w/those of modified techniques. I've read several articles suggesting a glycerol shock and/or adding chloroquine to the media.

My question is: which would be more beneficial for me to use? I am hesitant to try both at once b/c of cell toxicity.

I am transfecting A293T cells with a retroviral vector for infection of human neuroblastoma cells. Eventually, the plan is to grow up enough cells to do a tail vein injection into mice.

Does anyone have any experience with these techniques? I feel like a bit of a fuddy-duddy using CaPO4, but the Mirus reagent is just not cooperating with my cells (low %ages, not toxicity).

The following are the protocols I've gathered:

*4-6 hours after transfection reaction has been applied to cells, gently remove media and replace with 10% glycerol (or DMSO) for 2-3 minutes. Remove and feed with complete media (various references).

*1 hour before transfection, replace cDMEM with cDMEM + 25 mM chloroquine. Add transfection reaction to this media, and incubate o/n (JoVE).

*After "DNA absorption" (4 h again??) replace transfection reaction-containing media with cDMEM + 100 mM chloroquine. Change media after 2-5 hours (Luthman & Magnusson, 1983).

Any tips would be greatly appreciated. I'm going to try the Mirus reagent (TransIT1) again at the same time, as I'm still thinking that's my best bet.

Much appreciation,

porce

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I almost forgot!

Does anyone have experience comparing glycerol shock v. DMSO?

Thanks again!

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we use both chloroquine and DMSO shock; never tried glycerol

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